ASF is an acute, febrile, highly contagious infectious disease listed by the World Organization for Animal Health (WOAH) as a notifiable disease (1), with a morbidity and mortality rate as high as 100% in domestic pigs when it first occurred in China (2, 3). ASFV, the causative agent of ASF, belongs to the Asfivirus genus within the Asfarviridae family. It was first reported in 1921 in East Africa, and rapidly spread to other African countries (4). ASFV outbreak was first reported in China in 2018 (5, 6), and it caused the death of 1.193 million pigs by November 2021 (7).

The major transmission routes of ASFV include direct pig-to-pig contact or indirect contact with virus-contaminated objects, such as excretory materials (8, 9), feed (10), water (10, 11), and needles (2). To prevent ASFV diffusion and maintain the health of pig populations, a partitioned approach has been developed and proven effective. This approach involves improving the biosecurity level of pig farms to reduce the risk of ASF introduction, strengthening monitoring procedures for early detection, culling and removing positive groups to eliminate the risk, and implementing strict disinfection measures to eliminate pollution sources and interrupt transmission routes (12). However, with the emergence of mutant ASFV strains, further improvements in this strategy are necessary.

Aerosol transmission is another important route for ASFV spread. Aerosol transmission occurs when susceptible animals inhale pathogen-carrying particles with a diameter of less than 5 μm (13). Aerosols typically contain suspended solid or liquid particles in the air (13). While a study in 1977 showed ASFV transmission up to a distance of 2.3 meters in a confined space, no detection of ASFV in the air was reported (14). However, since 2012, air sampling methods have proven effective in detecting ASFV particles in the air. Although only a few experimental studies (15, 16) have reported aerosol transmission of ASFV, no field studies have been conducted to date. In this study, we present evidence that aerosols carrying ASFV can be found in piggeries under field conditions.

In this field study, we monitored the detection of ASFV in aerosol-associated samples in an ASFV-positive farm over a 24 day period following the confirmation of the first case of ASFV-positive pigs.

As shown in Figure 2, pigs of whole herds in Rooms A and B have been detected throughout the monitoring period, from Day 0 to Day 24. The Cq values of positive pigs were shown in Supplementary Table S1. In Room A, aerosol samples initially tested positive on Day 6, and continued to be positive until Day 24, despite negative samples on Days 12 and 15. Interestingly, Cq values of aerosol samples on Day 6 and 9 were lower compared to those on Day 18, 21, and 24, possibly indicating the removal of most ASFV-positive pigs in the later stage. Dust samples collected from air outlets were the last to be test positive on Day 9 among all sample types, and remained positive until Day 24. Furthermore, from Day 15 on, a downward trend in Cq values was observed from Day 15 onwards, suggesting the accumulation of the virus in the dust. These findings suggest that during an ASFV outbreak, ASFV particles excreted from infected pigs can be present in suspended aerosols and settling dust.

In Room B, as shown in Figure 2, ASFV-positive dust collected from air inlets was first detected on Day 15. Notably, there was a significant drop in the Cq value on Day 21. On the same day, pigs and aerosol samples were also detected as positive for ASFV, suggesting a possible association with the presence of positive dust in the air inlets.

Figure 2 also reveals that outdoor aerosol samples first tested positive on Day 9, coinciding with the collection of dust samples from air outlets in Room A. Additionally, outdoor aerosol samples remained positive until Day 24.

To investigate whether the dust in the air inlets of Room B originated from Room A, fluorescent powder was used to trace the dust trajectory from Room A. As depicted in Figure 3, 3 days later, fluorescent spots were observed on gauzes from both the air outlets in Room A and the air inlets in Room B, indicating the potential transmission of dust from Room A to Room B.

Aerosol transmission of infectious agents is widely recognized as one of the most challenging routes to prevent and control (19), particularly in commercial farms. Several swine virus, including foot-and-mouth disease virus (FMDV) (20), porcine reproductive and respiratory syndrome virus (PRRSV) (21), porcine epidemic diarrhea virus (PEDV) (22), and influenza A virus (IAV) (23), have been reported to spread through aerosols. However, the aerosol transmission of ASFV has been has been a subject of debate, with limited evidence from experimental studies (15, 16). Moreover, the current prevention strategies do not specifically address aerosol transmission risks. In this case study, we have found evidence of aerosol transmission of ASFV between two piggeries under field conditions.

By analyzing the dates of the first detection of different samples, we have identified a complete and clear chain of ASFV transmission via aerosols: infected pigs in Room A release aerosols, which contaminate the dust on air outlets in Room A. Subsequently, outdoor aerosols become contaminated, leading to the deposition of contaminated dust on air inlets in Room B, resulting in the transmission of aerosols and/or infected pigs to Room B. This represents a novel transmission route of ASFV between piggeries. The source of ASFV-positive aerosols is likely the excretions and secretions of ASFV-positive pigs, including urine, sneezes and feces (7). Previous research has proven that the positive aerosols were associated with viruses in feces (15), supporting our hypothesis. Dust also plays a crucial role in spreading ASFV particles among piggeries, although it is often overlooked by farmers due to its presence in hard-to-reach locations.

The presence of ASFV-positive dust poses a significant risk to the entire herd, especially if it becomes agitated due to factors such as sudden changes in wind direction or feeding activities in piggeries. In addition, contaminated feed, which has been identified as a primary risk for ASFV transmission (24, 25), can contribute to the presence of ASFV in dust. Therefore, timely removal of dust in piggeries is crucial for the control and prevention of ASFV transmission.

Outdoor aerosol detection is another critical factor to consider. Due to the wide range of outdoor aerosols, detecting their presence can be challenging. In our study, we employed the GR1356-Microbial concentration sampler, which allowed continuous aerosol collection for 6 h at a time. Outdoor aerosol samples remained positive until Day 24 during the experimental period, highlighting the persistent risk of aerosol transmission. Air filtration systems have been proven effective in preventing aerosol transmission of other pathogens, such as PRRSV (26). Therefore, integrating air filtration systems into the biosecurity measures against ASFV and other pathogens is recommended, especially for small farms with poor biosecurity practices in China (7).

The travel distance of viral aerosols is a significant concern. Wilkinson et al. demonstrated that ASFV can be transmitted through the air with a maximum distance of 2.3 meters (14). In our study, the distance between Rooms A and B was 10 meters, indicating that ASFV aerosols traveled at least 10 m. The difference in transmission distance could be attributed to environmental factors such as the outdoor temperature and the wind speed, as the temperature was below 4°C and strong winds were prevalent in northern China in winter. Furthermore, the transmission distance might also be influenced by the strain of the virus. In this case study, the ASFV strain belonged to Genotype I, causing mild onset of infection and chronic disease (27), and previous research has suggested that lower virulence strains tend to be highly transmissible (28). Further research is needed to investigate the transmission distance of ASFV aerosols.

In conclusion, this case study provides evidence of aerosol transmission of ASFV under field conditions, expanding our understanding of ASFV transmission routes. We emphasize the importance of considering air inlet and outlet filtration, strengthening air disinfection measures, and reducing dust levels in pig farms to create a low-risk environment with fresh air for pig herds.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

Ethical review and approval were not required for the animal study because the manuscript is a case study of spontaneous disease. Written informed consent was obtained from the owners for the participation of their animals in this study.

XL conceived and designed the analyzation method. XL, ZH, and MF collected and analyzed the data, and also wrote the original draft and reviewed and edited the manuscript. XT, WW, WG, LB, and XJ contributed to collect samples from field farms and the data of qPCR test. All authors contributed to the article and approved the submitted version.

This work was supported by Taishan Industry Leadership Talent Project of Shandong province in China, National Key R&D Program of China (2021ZD0113800) and the earmarked fund for CARS (CARS-35).

XL, ZH, MF, and WW were employed by Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd. XL, ZH, MF, XT, WG, LB, and XJ were employed by Shandong New Hope Liuhe Agriculture and Animal Husbandry Technology Co., Ltd. (NHLH Academy of Swine Research).

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